Thermo Scientific Competent Cells

Thermo Scientific DH5α competent cells

The transformation efficiencies of DH5α competent cells and 5-alpha competent E. coli (NEB) were compared using pUC19 DNA and following the manufacturers’ recommended protocols. The transformation efficiency specification for DH5α competent cells is 1.0 x 10⁹ CFU/µg DNA (represented by the red dashed line). The values shown are the averages of five transformations.

Thermo Scientific DH10B competent cell transformation efficiency in the CloneJET PCR cloning workflow

A 976 bp PCR product was generated using either Thermo Scientific DreamTaq Green PCR Master Mix (2X) (represented by grey bar) or Thermo Scientific Phusion Hot Start II DNA Polymerase (represented by blue bar) and ligated into a cloning vector according to the manufacturers’ protocols. The transformation efficiency obtained with the control fragment provided with the CloneJET PCR Cloning Kit is represented by the yellow bar. Ligation mixtures were used to transform Thermo Scientific DH10B cells. The expected transformation efficiency of competent cells is >1 x 10⁷ CFU/µg DNA. The transformation efficiency specification of DH10B competent cells with pUC19 DNA is 1 x 10⁹ CFU/µg DNA. The values shown are the average of five transformations. Error bars represent standard deviations.

The Thermo Scientific Phusion Site-Directed Mutagenesis Kit has been tested using the control plasmid and control primer mix provided in the kit, in conjunction with Thermo Scientific DH10B competent cells. The primer mix reverts the mutated internal stop codon to a functional codon; a successful mutagenesis reaction results in blue colonies on ampicillin agar plates containing X-Gal and IPTG. A high rate of mutagenesis and high transformation efficiency means that numerous mutants can be directly subjected to sequencing, eliminating additional analysis steps.